Cyclitol containing carbohydrates from human tissue which regulate glycogen metabolism

ABSTRACT

The application relates to the purification and characterisation of a family of P-type inositolphosphoglycans (IPGs) from human liver and placenta. These substances are shown to have P-type biological activity, e.g. activating pyruvate dehydrogenase (PDH) phosphatase. The characterisation of the compounds demonstrates that they contain metal ions, in particular Mn 2+  and/or Zn 2+ , and optionally phosphate. The compounds and their antagonists have uses as pharmaceuticals, e.g. for the treatment of diabetes, and in screening for synthetic analogues.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.09/254,748 (filed Jun. 14, 1999), which is a 371 of App. No.PCT/GB97/02533 (filed Sep. 11, 1997), which claims the benefit of UKApp. No. 96/18929.5 (filed Sep. 11, 1996).

FIELD OF THE INVENTION

[0002] The present invention relates to the characterisation of secondmessengers of insulin and other growth factors that regulate glycogenmetabolism. In particular, the present invention relates to substanceswhich are cyclitol containing carbohydrates, said substances containingMn²⁺ and/or Zn²⁺ ions, to cyclitol containing carbohydrates asobtainable from human liver or human placenta, to compositionscomprising these substances, and to the uses of these substances.

BACKGROUND OF THE INVENTION

[0003] Many of the actions of growth factors on cells are thought to bemediated by a family of inositol phosphoglycan (IPG) second messengers(T W Rademacher at al, Brazilian J. Med. Biol. Res., 27, 327-341,(1994)). It is thought that the source of IPGs is a “free” form ofglycosyl phosphatidylinositol (GPI) situated in cell membranes. IPGs arethought to be released by the action of phosphatidylinositol-specificphospholipases following ligation of growth factor to receptors on thecell surface.

[0004] There is evidence that IPGs immediate the action of a largenumber of growth factors including insulin, nerve growth factor,hepatocyte growth factor, insulin-like growth factor I (IGF-I),fibroblast growth factor, transforming growth factor β, the action ofIL-2 on B-cells and T-cells, ACTH signalling of adrenocortical cells,IgE, FSH and hCG stimulation of granulosa cells, thyrotropin stimulationof thyroid cells, cell proliferation in the early developing ear and ratmammary gland. However, to date, most of the research in this area hasconcentrated on the second messengers released by cells in response toinsulin. For example, insulin stimulates rapid hydrolysis ofmembrane-associated GPI molecules in myocytes, adipocytes, hepatomacells and T-cells. Recently, it has become clear that, at least whereinsulin is concerned, the released IPGs play an essential role as secondmessengers, and can in fact mimic many of the effects of insulin in theabsence of the hormone.

[0005] Soluble IPG fractions have been obtained from a variety of animaltissues including rat tissues (liver, kidney, muscle brain, adipose,heart) and bovine liver. IPG biological activity has also been detectedin malaria parasitized RBC and mycobacteria. The ability of ananti-inositolglycan antibody to inhibit insulin action on humanplacental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPGaction on rat diaphragm and chick ganglia suggests cross-speciesconservation of some three-dimensional features. However, it is wellestablished that species-specific glycoconjugates are a commoncharacteristic and structural characteristics determined on non-humanderived IPG may not be found on the human derived material.

[0006] We have divided the family of IPG second messengers into distinctA and P-type subfamilies on the basis of their biological activities. Inthe rat, release of the A and P-type mediators has been shown to betissue-specific (Kunjara et al, Biopolymers and Bioproducts: Structure,Function and Applications, J. Svast et al (ed), Dokya Publications,301-306, (1995)). Although in the past it has not been possible toisolate single purified components from the tissue derived IPGfractions, much less in sufficient quantities to allow structuralcharacterisation, there have been studies of the biological activitiesof the IPG containing fractions, and speculation as to the identity ofthe active components from non-human sources of the fractions based onindirect evidence from metabolic labelling and cleavage techniques.

[0007] Biological activity studies have shown that A-type mediatorsmodulate the activity of a number of insulin-dependent metabolic effectssuch as acetylCoA carboxylase (activates), cAMP dependent protein kinase(inhibits), adenylate cyclase (inhibits) and cAMP phosphodiesterases(stimulates). In contrast, P-type mediators modulate the activity ofenzymes such as pyruvate dehydrogenase phosphatase (stimulates) andglycogen synthase phosphatase (stimulates). The A-type mediators mimicthe lipogenic activity of insulin on adipocytes, whereas the P-typemediators mimic the glycogenic activity of insulin on muscle. Both A andP-type mediators are mitogenic when added to fibroblasts in serum freemedia. The ability of the mediators to stimulate fibroblastproliferation is enhanced if the cells are transfected with theEGF-receptor. A-type mediators can stimulate cell proliferation in chickcochleovestibular ganglia.

[0008] Despite these studies, evidence for the presence of a family ofsoluble IPG-type mediators in a primary target organ for insulin actionin humans has not yet been established. Further, research in this areahas been severely hampered by the limited availability of the A andP-type IPGs in fractions derived from mammalian tissues. In particular,there have been experimental difficulties in identifying, isolating andcharacterising the active components of the IPG fractions having A- andP-type biological activity.

[0009] Thus, studies on the measurement in urine of chiro and myoinositol have been complicated by the fact that both breakdown ofendogenous IPGs and dietary sources of the sugars will be present.Accordingly, prior art studies in this area which assumed that theP-type mediator contains chiro inositol and that the A-type mediatorcontains myo-inositol must be interpreted with caution, see Fonteles, MC, Huang, L C, Larner, J, Diabetologia, 39:731-734, (1996), in which theauthors report that they incorrectly identified the inositol in theP-type mediator which is pinitol and not chiro-inositol. As pinitol isnot converted to chiro-inositol by the acid conditions used incarbohydrate analysis, this is a case of misidentification.

[0010] Further, analysis of material isolated by metabolic labellingwith radionuclides or post-isolation labelling of extracted materialcannot be related to the chemical active substance, since one is onlyfollowing the labelled material and the actual active substance couldco-isolate but not be labelled. In addition, various enzymic or chemicaltreatments of the compounds used to determine structural characteristicsinactivate the compound making further structural steps impossible sinceone can no longer relate activity and structure. Further, as the activecomponents of the A- and P-type IPG fractions are believed to becarbohydrates rather than proteins, they cannot be produced byrecombinant DNA technology.

[0011] Thus, while there has been speculation in the art as to thechemical identity of these components, to date, there has been noisolation of an active component and no demonstration that it has A- orP-type biological activity.

SUMMARY OF THE INVENTION

[0012] Our purification reported here from human tissues generates anon-radiolabelled compound which can be visualised on Dionexchromatography and by mass spectrometry. In rats, we can relate changesin the amount of compound present to the insulin stimulation of thetissue. As the rat compounds were isolated by the same protocol as thatused to isolate the human compounds, by analogy, the human substancesdescribed here are released in response to insulin stimulation. Thisdefines them as insulin-responsive compounds. We have also purifiedP-type fractions using Vydac HPLC chromatography and shown that thecompounds obtained have P-type biological activity.

[0013] Broadly, the present invention is based on the isolation of anactive component of a P-type fraction derived from human liver orplacenta in sufficient quantity to characterise this P-type substancefor the first time. In particular, this characterisation showed thatthis substance contains metal ions, in particular Mn²⁺ and/or Zn²⁺, andhas a biological activity associated with P-type IPG fractions, namelythe property of activating pyruvate dehydrogenase (PDH) phosphatase.

[0014] Accordingly, in one aspect, the present invention provides aP-type substance which is a cyclitol containing carbohydrate, saidsubstance containing Mn²⁺ and/or Zn²⁺ ions and optionally phosphate.This finding was made as some P-type fractions isolated using Vydac HPLCchromatography did not contain phosphate but were biologically active,indicating that phosphate is not essential for biological activity.

[0015] Accordingly, in the present application, references to“inositolphosphoglycans” or “IPGs” include compounds in which phosphateis not present. These compounds are alternatively be termedinositolglycans (IGs).

[0016] We have further found the P-type substance to have the followingproperties:

[0017] 1. Migrates near the origin in descending paper chromatographyusing 4/1/1 butanol/ethanol/water as a solvent.

[0018] 2. Some of the P-type substances contain phosphate which isdirectly related to activity.

[0019] 3. The free GPI precursors are resistant to cleavage by GPI-PLCfrom bacterial sources.

[0020] 4. They are partially retained on C-18 affinity resin (Table 1)

[0021] 5. They are bound on Dowex AG50 (H+) cation exchange resin (Table1).

[0022] 6. They are bound on an AG3A anion exchange resin (Table 1).

[0023] 7. The activity is resistant to pronase.

[0024] 8. They are detected using a Dionex chromatography system orVydac HPLC chromatography (see FIGS. 7 to 9).

[0025] The substance may also have one or more of the followingactivities associated with P-type IPG fractions:

[0026] (a) stimulates the activity glycogen synthase phosphatase;

[0027] (b) mitogenic when added to fibroblasts in serum free medium;

[0028] (c) stimulates pyruvate dehydrogenase phosphatase.

[0029] Thus, while the prior art discloses that the biologicalactivities associated with P-type IPG can be detected in fractionsobtained from bovine and rat tissues, it does not isolate orcharacterise the component from the fraction and demonstrate that it hasa P-type IPG biological activity.

[0030] In a further aspect, the present invention provides a substancewhich is a cyclitol containing carbohydrate, said substance containingMn²⁺ and/or Zn²⁺ ions and optionally phosphate, as obtainable by fromhuman liver or placenta by:

[0031] (a) making an extract by heat and acid treatment of a liverhomogenate, the homogenate being processed from tissue immediatelyfrozen in liquid nitrogen;

[0032] (b) after centrifugation and charcoal treatment, allowing theresulting solution to interact overnight with an AG1-X8 (formate form)anion exchange resin;

[0033] (c) collecting a fraction having P-type IPG activity obtained byeluting the column with 10 mM HCl;

[0034] (d) neutralising to pH 4 (not to exceed pH 7.8) and lyophilisingthe fraction to isolate the substance;

[0035] (e) descending paper chromatography using 4/1/1butanol/ethanol/water as solvent;

[0036] (f) purification using high-voltage paper electrophoresis inpyridine/acetic acid/water; and,

[0037] (g) purification using Dionex anion exchange chromatography, orpurification and isolation using Vydac HPLC chromatography to obtain theisolated P-type substance.

[0038] In a further aspect, the present invention provides an isolatedsubstance which is a P-type cyclitol containing carbohydrate comprisingMn²⁺ and Zn²⁺ ions and has the biological activity of activatingpyruvate dehydrogenase (PDH) phosphatase, wherein the substance has amolecular weight determined using negative mode MALDI mass spectroscopyas shown in FIG. 11, or a molecular weight related to one of themolecular weights set out in FIG. 11 by the addition or subtraction ofone or more 236 m/z structure units.

[0039] In a further aspect, the present invention providespharmaceutical compositions comprising a P-type substance as describedabove, optionally in combination with insulin or an A-type substance forsimultaneous or sequential administration. These compositions can beused in the treatment of disorders in which the glycogenic activity ofpatient has in some way been impaired, e.g. in obese NIDDM patients whodo not produce enough P-type IPG, compared to A-type production, i.e.the ratio of A- and P-type IPGs in these patients is out of balance.

[0040] A detailed discussion on the cell signalling arrangements and therelease of IPG second messengers in response to growth factors isprovided in our co-pending International patent application numberPCT/GB96/00669 filed on Mar. 20, 1996.

[0041] In a further aspect, the present invention provides antagoniststo the substances described above and pharmaceutical compositionscomprising these antagonists. These compositions can be useful in thetreatment of conditions in which P-type IPGs are overproduced and/or toantagonise one of the activities of the P-type IPGs. Such an antagonistmay be a related IPG which is able to compete with the P-type IPG buthave no biological action in its own right. For example, thedephosphorylated form of the P-type IPG which has reduced bioactivitycould compete with the active phosphorylated P-type for uptake into thecell.

[0042] In a further aspect, the present invention provides a substanceor antagonist as described above for use in a method of medicaltreatment.

[0043] It is expected that synthetic compounds containing all or part ofthe active substituents of the P-type IPG could be useful astherapeutics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 shows DX500 HPLC of purified P-type family of IPG mediatorsfrom human liver.

[0045]FIG. 2 shows the stimulation of EGFR T17 fibroblasts and PDHphosphatase. Panel A: Serial dilutions of stock human liver derivedtype-A and type-P were assayed for their ability to stimulateproliferation. Control represents the proliferation of the fibroblastsin serum-free medium with addition of IPG. Panel B: Stimulation ofbovine heart derived PDH phosphatase was linear for both human and ratderived type-P mediator. The amount of mediator used was volume of stock(see Materials and Methods).

[0046]FIG. 3 shows the purification of IPG by descending paperchromatography. Descending paper chromatography profiles of control- andpronase E-treated IPG type-A and type-P, panels A and B respectively,following analysis for phosphate content. Panels C and D show the freeamino groups analysis in the same chromatographic fractions. Forclarity, only the first 10 fractions are displayed in each panel forpronase treated samples. The profiles for untreated mediators wasidentical. The solvent front was +35 cm.

[0047]FIGS. 4a and 4 b show the high voltage electrophoresis of IPGtype-A and type-P indicators. FIG. 4a shows a representativeelectrophoretogram of IPG type-P (black) HVE following detection ofphosphate. FIG. 4b shows the effect of selected fractions of IPG type-Pon cell proliferation after pronase treatment, descending paperchromatography and HVE purification steps. FIG. 4b shows the effect ofcrude preparation of IPG type-A and IPG type-P at a final dilution of1/80 on [³H]thymidine incorporation into EGFR T17 fibroblasts. Themigration positions of bromophenol blue (BB), inositol monophosphate(IP1) and inositol di/tri-phosphate are indicated by arrows.

[0048]FIG. 5 shows the correlation between [³H]thymidine incorporation,PDH phosphatase stimulating activity and phosphate content of selectedfractions from the HVE electrophoretogram. Panel A: HVE fractions wereassayed for their ability to stimulate PDH phosphatase. The correlationbetween both effects was r=0.97. Panel B shows the correlation (r=0.87)between phosphate content and the stimulation by the same fractions of[³H]thymidine incorporation into EGFR T17 fibroblasts.

[0049]FIG. 6 shows the quantitative increase in IPG release followinginfusion with insulin.

[0050]FIG. 7 the family of IPGs responsive to insulin as detected byDX500 anion exchange chromatography. Peaks with * are not present in thepre-insulin stimulated rat liver.

[0051]FIG. 8 shows the phosphate content of a family of P-typesubstances isolated and purified using Vydac HPLC chromatography.

[0052]FIG. 9 shows the bioactivity of selected P-type substancesisolated and purified using Vydac HPLC chromatography.

[0053]FIG. 10 shows the Dionex peak of a P-type fraction isolated usingVydac HPLC chromatography, showing that this fraction corresponds topeak 23 shown in FIGS. 1 and 7.

[0054]FIG. 11 shows a MALDI mass spectrum (negative mode) of a family ofP-type IPGs.

DETAILED DESCRIPTION

[0055] Mimetic Design

[0056] The designing of mimetics to a known pharmaceutically activecompound is a known approach to the development of pharmaceuticals basedon a “lead” compound. This might be desirable where the active compoundis difficult or expensive to syrthesise or where it is unsuitable for aparticular method of administration, e.g. peptides are unsuitable activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis and testingis generally used to avoid randomly screening large number of moleculesfor a target property.

[0057] There are several steps commonly taken in the design of a mimeticfrom a compound having a given target property. Firstly, the particularparts of the compound that are critical and/or important in determiningthe target property are determined. These parts of the compoundconstituting its active region are known as its “pharmacophore”.

[0058] Once the pharmacophore has been found, its structure is modelledto according its physical properties, e.g. stereochemistry, bonding,size and/or charge, using data from a range of sources, eg spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

[0059] In a variant of this approach, the three-dimensional structure ofthe ligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

[0060] A template molecule is then selected onto which chemical groupswhich mimic the pharmacophore call DC grafted. The template molecule andthe chemical groups grafted on to it can conveniently be selected sothat the mimetic is easy to synthesise, is likely to bepharmacologically acceptable, and does not degrade in vivo, whileretaining the biological activity of the lead compound. The mimetic ormimetics found by this approach can then be screened to see whether theyhave the target property, or to what extent they exhibit it. Furtheroptimisation or modification can then be carried out to arrive at one ormore final mimetics for in vivo or clinical testing.

[0061] In the present case, it is expected that synthetic compoundscontaining all or part of the active substituents of the P-type IPGcould be useful as therapeutics.

[0062] Antagonists

[0063] Antagonists to the P-type substances include substances whichhave one or more of the following properties:

[0064] (a) substances capable of inhibiting release of the P-typemediators;

[0065] (b) substances capable of reducing the levels of P-type mediatorsvia a binding substance (e.g. an antibody or specific binding protein);and/or,

[0066] (c) substances capable of reducing the effects of P-typemediators.

[0067] In one embodiment, the IPG antagonists are specific bindingproteins. Naturally occurring specific binding proteins can be obtainedby screening biological samples for proteins that bind to IPGs.

[0068] In a further embodiment, the antagonists are antibodies capableof specifically binding to P-type IPGs. The production of polyclonal andmonoclonal antibodies is well established in the art. Monoclonalantibodies can be subjected to the techniques of recombinant DNAtechnology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP-A-184187, GB-A-2188638or EP-A-239400. A hybridoma producing a monoclonal antibody may besubject to genetic mutation or other changes, which may or may not alterthe binding specificity of antibodies produced.

[0069] Antibodies may be obtained using techniques which are standard inthe art. Methods of producing antibodies include immunising a mammal(e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the proteinor a fragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and screened,preferably using binding of antibody to antigen of interest. Forinstance, Western blotting techniques or immunoprecipitation may be used(Armitage et al, Nature, 357:80-82, 1992). Isolation of antibodiesand/or antibody-producing cells from an animal may be accompanied by astep of sacrificing the animal.

[0070] As an alternative or supplement to immunising a mammal with apeptide, an antibody specific for a protein may be obtained from arecombinantly produced library of expressed immunoglobulin variabledomains, e.g. using lambda bacteriophage or filamentous bacteriophagewhich display functional immunoglobulin binding domains on theirsurfaces; for instance see WO92/01047. The library may be naive, that isconstructed from sequences obtained from an organism which has not beenimmunised with any of the proteins (or fragments), or may be oneconstructed using sequences obtained from an organism which has beenexposed to the antigen of interest.

[0071] Antibodies according to the present invention may be modified ina number of ways. Indeed the term “antibody” should be construed ascovering any binding substance having a binding domain with the requiredspecificity. Thus the invention covers antibody fragments, derivatives,functional equivalents and homologues of antibodies, including syntheticmolecules and molecules whose shape mimics that of an antibody enablingit to bind an antigen or epitope.

[0072] Example antibody fragments, capable of binding an antigen orother binding partner are the Fab fragment consisting of the VL, VH, Cland CHl domains; the Fd fragment consisting of the VH and CHl domains;the Fv fragment consisting of the VL and VH domains of a single arm ofan antibody; the dAb fragment which consists of a VH domain; isolatedCDR regions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

[0073] Humanised antibodies in which CDRs from a non-human source aregrafted onto human framework regions, typically with the alteration ofsome of the framework amino acid residues, to provide antibodies whichare less immunogenic than the parent non-human antibodies, are alsoincluded within the present invention

[0074] A hybridoma producing a monoclonal antibody according to thepresent invention may be subject to genetic mutation or other changes.It will further be understood by those skilled in the art that amonoclonal antibody can be subjected to the techniques of recombinantDNA technology to produce other antibodies or chimeric molecules whichretain the specificity of the original antibody. Such techniques mayinvolve introducing DNA encoding the immunoglobulin variable region, orthe complementarity determining regions (CDRs), of an antibody to theconstant regions, or constant regions plus framework regions, of adifferent immunoglobulin. See, for instance, EP-A-184187, GB-A-2188638or EP-A-0239400. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023.

[0075] hybridomas capable of producing antibody with desired bindingcharacteristics are within the scope of the present invention, as arehost cells, eukaryotic or prokaryotic, containing nucleic acid encodingantibodies (including antibody fragments) and capable of theirexpression. The invention also provides methods of production of theantibodies including growing a cell capable of producing the antibodyunder conditions in which the antibody is produced, and preferablysecreted.

[0076] The antibodies described above may also be employed in thediagnostic aspects of the invention by tagging them with a label orreporter molecule which can directly or indirectly generate detectable,and preferably measurable, signals. The linkage of reporter moleculesmay be directly or indirectly, covalently, e.g. via a peptide bond ornon-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule.

[0077] One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

[0078] Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

[0079] In a further embodiment, the IPG antagonists are syntheticcompounds. These may be produced by conventional chemical techniques orusing combinatorial chemistry, and then screened for IPG antagonistactivity. These compounds may be useful in themselves or may be used inthe design of mimetics, providing candidate lead compounds fordevelopment as pharmaceuticals. Synthetic compounds might be desirablewhere they are comparatively easy to synthesize or where they haveproperties that make them suitable for administration aspharmaceuticals, e.g. antagonist which are peptides may be unsuitableactive agents for oral compositions if they are degraded by proteases inthe alimentary canal. Mimetic design, synthesis and testing is generallyused to avoid randomly screening large number of molecules for a targetproperty.

[0080] Pharmaceutical Compositions

[0081] The mediators and antagonists of the invention can be formulatedin pharmaceutical compositions. These compositions may comprise, inaddition to one or more of the mediators or antagonists, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialmay depend on the route of administration, e.g. oral, intravenous,cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0082] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may include a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

[0083] For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, isotonic vehicles such as Sodium Chloride Injection,Ringer's Injection, Lactated Ringer's Injection. Preservatives,stabilisers, buffers, antioxidants and/or other additives may beincluded, as required.

[0084] Whether it is a polypeptide, antibody, peptide, small molecule orother pharmaceutically useful compound according to the presentinvention that is to be given to an individual, administration ispreferably in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc., is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.In a preferred embodiment, dosage levels will be determined as producingeuglycaemic conditions.

[0085] A composition may be administered alone or in combination withother treatments, either simultaneously or sequentially dependent uponthe condition to be treated.

[0086] Diagnostic Methods

[0087] Methods for determining the concentrations of analytes inbiological samples from individuals are well known in the art and can beemployed in the context of the present invention to determine the ratioof P- and A-type inositolphosphoglycans (IPGs) in a biological samplefrom a patient. This in turn can allow a physician to determine if theratio or level of P- and A-type IPGs is out of balance having regard tothe patient and the condition being tested for. Examples of diagnosticmethods are described in the experimental section below.

[0088] Preferred diagnostic methods rely on the determination of theratio of P- and A-type IPGs. The methods can employ biological samplessuch as blood, serum, tissue samples or urine.

[0089] The assay methods for determining the concentration of P- andA-type IPGs typically employ binding agents having binding sites capableof specifically binding to one or more of the P- or A-type IPGs inpreference to other molecules. Examples of binding agents includeantibodies, receptors and other molecules capable of specificallybinding the IPGs. Conveniently, the binding agent(s) are immobilised onsolid support, e.g. at defined locations, to make them easy tomanipulate during the assay.

[0090] The sample is generally contacted with the binding agent(s) underappropriate conditions so that P-and A-type IPGs present in the samplecan bind to the binding agent(s). The fractional occupancy of thebinding sites of the binding agent(s) can then be determined using adeveloping agent or agents. Typically, the developing agents arelabelled (e.g. with radioactive, fluorescent or enzyme labels) so thatthey can be detected using techniques well known in the art. Thus,radioactive labels can be detected using a scintillation counter orother radiation counting device, fluorescent labels using a laser andconfocal microscope, and enzyme labels by the action of an enzyme labelon a substrate, typically to produce a colour change. The developingagent(s) can be used in a competitive method in which the developingagent competes with the analyte for occupied binding sites of thebinding agent, or non-competitive method, in which the labelleddeveloping agent binds analyte bound by the binding agent or to occupiedbinding sites. Both methods provide an indication of the number of thebinding sites occupied by the analyte, and hence the concentration ofthe analyte in the sample, e.g. by comparison with standards obtainedusing samples containing known concentrations of the analyte. Inpreferred embodiments, this can then be used to determine the P:A typeratio.

[0091] Methods

[0092] Isolation and Characterisation of Inositol Phosphoglycans.

[0093] Inositolphosphoglycans (IPG) were purified as follows from frozenhuman liver. The frozen tissue (90 g) was powdered under liquid nitrogenand placed directly into boiling 50 mM formic acid containing 1 mM EDTAand 1 mM 2-S mercaptoethanol (3 mL of buffer per gram (wet weight) oftissue). After 1 min homogenisation with a polytron mixer (Kinematica,Littau, Switzerland), the solution was further boiled for 5 min. Thesolution was then cooled on ice and centrifuged at 29,500 g for 2 h at4° C. The supernatant was treated with 10 mg/mL activated charcoal for30 min with stirring at 4° C. The charcoal suspension was centrifuged at29,500 g for 1 h at 4° C. and the clear supernatant recovered. Thesolution was then diluted ten-fold with distilled water, adjusted to pH6.0 with 10% NH₄OH solution and then gently shaken overnight at roomtemperature with AG1-X8 (formate form) resin (0.3 mL resin per mLsolution). The resin was then poured into a chromatography column(2.5×60 cm) and washed sequentially with water (2 bed volumes) and 1 mMHCl (2 bed volumes). Then, the material was eluted with 10 mM HCl (5 bedvolumes) to obtain an IPG P-type fraction. This fraction was adjusted topH 4.0 with 10% NH₄OH solution and then dried in a rotary evaporator.The dried material was redissolved in distilled water, lyophilised twiceand divided into five aliquots for both chemical and biochemicalanalyses. For analyses, aliquots of each type of preparation weredissolved in 200 μL of Hanks Medium and adjusted to pH 7.0 with 1 M KOH.The mediators extracted from the equivalent of 16 g (wet weight) oftissue were dissolved in a final volume of 200 μL (stock solution).Therefore, 10 μL of stock represents the amount of the P-type mediatorrecovered from 800 mg of starting tissue.

[0094] Pronase Treatment.

[0095] IPG was treated with Pronase E as described elsewhere. Briefly, astock solution of the enzyme (10 mg/mL) was preincubated at 60° C. for30 min in 100 mM Tris-HCl buffer, pH 8.0, to inactivate contaminatingenzymes which may be present. Digestion of the sample was started byaddition of pronase solution (30 μL) to IPG samples in 200 μL of 100 mMTris-HCl buffer at pH 7.8 at 37° C. After two hours, the reaction wasterminated by boiling for 3 min and removed by acid precipitation.

[0096] IPG Purification by Paper Chromatography.

[0097] IPG was dissolved in a minimum amount of water and applied to a 3MM chromatography paper (3×50 cm, origin at 8.5 cm). Descending paperchromatography was performed using n-butanol/ethanol/water (4:1:1,v/v/v) and the chromatogram was developed for 9 h. After drying, thepaper was cut every centimetre (−1 to +35 cm from the origin) and thematerial associated the fraction eluted with water (60 μL, 5 washes).The fraction was evaporated to dryness and redissolved either in wateror in Hanks solution (60 μL) and neutralized with 1 N KOH prior to thedetermination of free amino groups, phosphate content or to assaybiological activities.

[0098] High Voltage Paper Electrophoresis.

[0099] The material eluted from fractions 1 to 6 after paperchromatography, was pooled, redissolved in a small volume of water andapplied to a 3 MM electrophoresis paper. Bromophenol blue and tritiatedinositol phosphates mixture were added as standards. The samples wereelectrophoresed for 30 min at 80 Vcm⁻¹ in pyridine/acetic acid/water(3:1:387, v/v/v), pH 5.4. Neutral compounds remained at the point ofapplication, while negatively charged compounds moved towards the anode.After the paper was dried, fractions were cut out every one cm andeluted with water.

[0100] Vydac HPLC Chromatography

[0101] This technique was used to isolate and purify individualfractions containing the mediators. The P-type IPG was applied to aVydac 301 PLX575 HPLC column. The column was eluted as follows: Solvent:Ammonium acetate 500 mM pH 5.5, Gradient conditions:  0-5% over 12minutes,  5-21% over the next 13 minutes, 21-80% over 25 minutes,80-100% over 5 minutes.

[0102] The fractions were then assayed for phosphate and growthpromoting activity using EGF-transfected fibroblasts.

[0103] Determination of Free Amino Groups.

[0104] Measurement of free amino groups was performed as describedbelow. Samples and standards (0-100 mmol of D-(+)-glucosaminehydrochloride, Sigma) were dissolved in ultrapure water (50 μL) beforesequentially adding sodium borate (0.14 M, pH 9) and fluorescamine (0.75mg/ml prepared in dry acetone). Emission fluorescence at 475 nm wasobserved after excitation at 390 nm using a spectrofluorimeter.

[0105] Determination of Phosphate Content.

[0106] Total phosphate levels were assayed as described below Samplesand standards (0-100 nmoles of Na₂HPO₄) were evaporated to dryness andhydrolysed with perchloric acid (70%) at 180° C. for 30 min. Aftercooling to room temperature, ultrapure water (250 μL), (NH₄)₂MoO₄ (100μL of a 2.5% solution) and ascorbic acid (100 μl of a 10% solution) weresequentially added. Colour development was achieved by heating thesamples at 95° C. for 15 min. Optical absorbance was measured at 655 nmin a microplate reader.

[0107] Interaction of IPG With Ion Exchange Resins and Sep-Pak C18Cartridges.

[0108] Thirty microliters of stock solution (see above) were loaded ontocolumns containing 600 μL of either AG3-X4 (HO⁻), AG5O-X12 (H⁺) or ontoSep Pack C18 cartridges and then eluted with water (5 bed volumes). Thesolutions were concentrated to dryness and the residues obtainedre-dissolved in 30 μL of Hanks and adjusted to pH 7.0.

[0109] Evaluation of cAMP-Dependent Protein Kinase Activity.

[0110] The ability of the IPG fraction to inhibit the activity of thecyclic AMP-dependent protein kinase was assessed by using histone IIA assubstrate. The reaction mixture (100 μL) contained 25 mM HEPES buffer(pH 7.6), 10 μM MgATP (10⁶ cpm [γ-³²P]ATP), histone IIA (50 μg protein),and catalytic subunit of PKA (60 units/mL). In all the determinations,10 μL of IPG solution (see above) was added to the reaction mixture.After incubation at 37° C. for 10 min, the reaction was stopped andproteins precipitated with 10% trichloroacetic acid (100 μL) and 2%bovine serum albumin (10 μL) and the incorporation of ³²P into proteinswas determined.

[0111] Evaluation of the Pyruvate Dehydrogenase Phosphatase (PDH)Activity.

[0112] Pyruvate dehydrogenase complex (PDC) and the PDH phosphatase wereprepared and stored at −80° C. until use. The assay for both PDHphosphatases, in the presence of absence of insulin mediator, was basedupon the initial rate of activation of inactivated, phosphorylated PDHcomplex. The initial activity of the PDC was 8-13 units/ml (1 unit ofenzyme produces 1 μmol NADH/min) and after inactivation with ATP,0.3-0.5 units/ml (inactivated PDC). A two stage assay was used toquantitate the phosphatase activity. A sample of inactivated PDC (50 μL)was preincubated at 30° C. with 1 mg/mL fat-free BSA, 10 mM MgCl₂, 0.1mM CaCl₂ and 1 mum DTT in 20 mM potassium phosphate buffer at pH 7.0(total volume 250 μL) for three minutes. At this time, 10 μL of the PDHphosphatase and 10 μL of IPG were added and the incubation continued fora further 2 min. At the end of this time, 200 μL of the mix was removedand added to 100 μL of 300 mM NaF. The activated PDH was determined atthe second stage photometrically by measuring the rate of production ofNADH. One hundred microliters of the stopped reaction were added to 1 mLof reaction mixture containing 50 mM potassium phosphate buffer at pH8.0, 2.5 mM β-NAD⁺, 0.2 mM TPP, 0.13 mM coenzyme A, 0.32 mM DTT and 2 mMsodium pyruvate. The production of NADH was followed at 340 nm for 5min.

[0113] Evaluation of Glycogen Synthase Activity

[0114] Glycogen synthase activity was assayed as follows. Fat cells wereprepared from epididymal adipose tissue using 140-150 g rats (Wistarstrain). Fat pads from 3 rats were cut into small pieces and incubatedin 8 ml of Krebs ringer bicarbonate containing 2% albumin plus 24 mg ofcollagenase for 25 minutes at 37° C. The isolated cells were filteredand washed in 3×10 ml Krebs ringer phosphate medium containing 30 mg/mlof bovine serum albumin (Fraction v, Sigma), the pH was adjusted to 7.4with NaOH after addition of albumin. The washed cells were alsoresuspended in Krebs ringer phosphate (10 ml of medium/g of originaltissue).

[0115] For measurement of enzyme activities, three incubation vesselswere set up, each contained 5 ml cells. There were no additions to thefirst, the second contained 20 μl insulin (25 mU/ml) and the third 10 μlplacenta IPG. Incubation was for 5 min in air. The tubes were then spunat 1500 rpm for 30 seconds and the supranatant discarded.

[0116] The reaction was stopped with 0.5 ml cold buffer (100 mM KF/10 mMEDTA/1 mM benzamidine (4), pH 7.0) and the cells homogenized andtransferred to Eppendorf tubes. These were spun at 10,000 g for 15 minand the middle layer collected for enzyme assay all tubes were set up induplicate and measured in the presence of 0.01 mM glucose 6-phosphateexcept the maximally activated glycogen synthase tube which contained7.2 mM glucose 6-phosphate. Samples (30 μl) were added to 60 μl of asolution containing 50 mM tris buffer (pH 7.8), 20 mM EDTA, 25 mM KF, 10mg/ml of glycogen, and 6.7 mM UDP-[U-¹⁴C] glucose (approximately 200,000cpm) and then incubated at 30° C. for 15 min. Glycogen synthase activityis expressed as a percentage of the total synthase activity (G6Pmaximum).

[0117] Measurement of Cellular Proliferation in Fibroblasts.

[0118] EGFRT17 fibroblasts were routinely grown in Dulbecco's ModifiedEagle's Medium (DMEM) containing 10% v/v foetal calf serum, 2 mML-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin at 37°C. in a humidified atmosphere of 5% CO₂. The cells were subcultured whenthey approached 80% confluence. The EGFRT17 cells are NIH 3T3fibroblasts transfected with the human epidermal growth factor receptor[32, 35]. To evaluate fibroblast cell proliferation, cells were platedinto 96-well microtitre wells at a density of 10⁴ cell per well in DMEMcontaining 100 FCS. After 24 h the medium was removed, the cells washedtwice with Hanks medium, serum free medium was added, and the cells wereincubated for a further 24 h period. At this point the cells werestimulated with serum, IPG preparations or the appropriate controls.Eighteen hours later [³H]thymidine (l μCi/well), the was added to eachwell for 4 h. At the end of this treatment, the cells were washed twicewith Hanks solution, trypsinised, and radioactivity associated withcellular DNA determined using a cell harvester. For the cellproliferation assays, the dilutions are final dilutions. For example,2.5 μL of the stock solution is added to a final volume of 100 μL, or1/40 dilution.

[0119] Protocol for Sandwich ELISA.

[0120] The protocol below sets out an indirect, non-competitive,solid-phase enzyme immunoassay (sandwich ELISA) for the quantificationof inositolphosphoglycans (IPG) in biological fluids, such as humanserum.

[0121] In the assay, monoclonal IgM antibodies are immobilised on asolid phase. Tissue culture supernatant, ascitic fluid from mice with aperitoneal tumour induced by injecting hybridoma cells into theperitoneum and purified monoclonal antibody have been used in theimmunoassay. F96 Maxisorp Nunc-Immuno plates were used for these assays.Maxisorp surface is recommended where proteins, specially glycoproteinssuch as antibodies, are bound to the plastic.

[0122] The immobilised antibody captures the antigen from the testsample (human serum or IPG used like a positive control).

[0123] A bridging antibody (a purified polyclonal IPG antibody fromrabbit) is needed to link the anti-antibody biotinylated to the antigen.

[0124] The detection method employs an anti-rabbit Ig, biotinylatedspecies-specific whole antibody (from donkey) and astreptavidin-biotinylated horseradish peroxidase complex (Amersham),ABTS and buffer for ABTS (Boehringer Mannheim).

[0125] The ELISA assay can be carried out as follows:

[0126] 1. Add 100 μl/well in all the steps.

[0127] 2. Add monoclonal antibody diluted 1:100 in PBS in a F96 MaxisorpNunc-Immuno plate. Incubate at least 2 days at 4° C.

[0128] 3. Wash with PBS three times.

[0129] 4. Add a blocking reagent for ELISA (Boehringer Mannheim) indistilled water (1:9) 2 hours at room temperature.

[0130] 5. Wash with PBS-Tween 20 (0,1%) three times.

[0131] 6. Add a purified polyclonal antibody (diluted 1:100 in PBS),overnight at 4° C.

[0132] 7. Wash with PBS-Tween 20 (0.1%) three times.

[0133] 8. Add an anti-rabbit Ig, biotinylated species-specific wholeantibody (from donkey) (Amersham) diluted 1:1000 in PBS, 1 h 30 min atroom temperature.

[0134] 9. Wash with PBS-Tween 20 (0.1%) three times.

[0135] 10. Add a streptavidin-biotinylated horseradish peroxidasecomplex (Amersham) diluted 1:500 in PBS, 1 h 30 min at room temperature.

[0136] 11. Wash with PBS three times.

[0137] 12. Add 2.2-Azino-di-(3-ethylbenzthiazoline sulfonate (6))diammonium salt crystals (ABTS) (Boehringer Mannheim) to buffer for ABTS(BM): Buffer for ABTS is added to distilled water (1:9 v/v). 1 mg ofABTS is added to 1 ml of diluted buffer for ABTS.

[0138] 13. Read the absorbance in a Multiscan Plus P 2.01 using a 405 mmfilter in 5-15 min.

[0139] Results

[0140] IPG Isolation and Biological Activities.

[0141] IPGs were extracted from human liver and placenta as follows.Briefly, the extract was prepared by heat and acid treatment of a tissuehomogenate, processed from tissue immediately frozen in liquid nitrogenafter removal from the patient, therefore preventing the action ofphosphatases. After centrifugation and charcoal treatment, the solutionwas allowed to interact overnight with an anion exchange resin (AG1-X8,formate form). The resin was washed sequentially with water and dilutehydrochloric acid. Elution with 10 mM HCl produced a P-type IPG fractionwhich was active in stimulating pyruvate dehydrogenase phosphatase andglycogen synthesis and stimulated proliferation of EGF receptortransfected fibroblast. This fraction was then subjected to descendingpaper chromatography using 4/1/1 butanol/ethanol/water as solvent,followed by purification using high-voltage paper electrophoresis inpyridine/acetic acid/water, and finally purification using Dionex.(trade mark) anion exchange chromatography. FIG. 1 shows a sharp spike(fraction no. 10) representing the major P-type substance.

[0142] Metal Analysis

[0143] Metal ion analysis was performed on a DX500 system with visibledetection at 520 nm.

[0144] The separation was achieved using IonPac mixed bed ion exchangecolumns with pyridine-2,6-dicarboxylic acid eluent and post columnreaction with pyridial azo resourcinol.

[0145] The P-type samples (P1 and P2) were reconstituted in 100 μl ofwater. 10 μl of this solution was taken, 10 μl of conc HCl added and thesample left overnight. Then, 80 μl of water was added to the mixture and10 μl of this solution was analysed. Blank HCl samples acted ascontrols. μg/ml Zn μg/ml Mn μg/ml Fe Averages P1 #1 12.31  3.10 14.042.27 μg/ml Zn #2 2.22 3.17 14.10 3.14 μg/ml Mn 14.07 μg/ml Fe P2 #1 2.493.35 15.28 2.45 μg/ml Zn #2 2.38 3.41 15.49 3.36 μg/ml Mn #3 2.47 3.3315.10 15.29 μg/ml Fe Blank #1 — — 10.24 #2 — — 10.26 10.28 μg/ml Fe #3 —— 10.33 Blank #1 — —  8.42 8.32 μg/ml Fe #2 — —  8.21

[0146] This shows for the first time that the P-type IPG isolated fromhuman liver contains Mn²⁺ and/or Zn²⁺ ion.

[0147] Inhibition of cAMP Dependent Protein Kinase.

[0148] The ability of the IPG to inhibit cAMP-dependent protein kinasewas tested by determining the incorporation of ³²P into washed histoneIIA. The addition of the P-type IPG fraction diluted 1/10, caused56.7±16.4 (n=4) percent inhibition of the kinase activity. While thiseffect was dose dependent, the P-type IPG did not give significantinhibition of kinase activity. These experiments are in agreement withour previous data for rat-derived IPGs. In contrast to the P-type IPGfraction, the A-type fraction we have described elsewhere contains thepredominant inhibitor activity against cAMP dependent protein kinase.

[0149] Stimulation of PDH Phosphatase.

[0150] The ability of the P-type IPG fraction to stimulate pyruvatedehydrogenase phosphatase isolated from bovine heart was determined.Table 1 shows that both human livers contained substantial PDHphosphatase stimulating activity in the P-type eluate. The amount ofactivity recovered was similar to that recovered from rat liver in theabsence of insulin stimulation (see Table 1). Insulin stimulation of ratliver results in a two-fold increase in activity 2 minutes after insulininfusion (see FIG. 6). We have previously used the nomenclature P-typeto denote the IPG family of compounds present in the 10 mM fraction, tohighlight this activity.

[0151] IPG can Substitute for Mn²⁺ In Vitro

[0152] The P-type insulin IPG can completely substitute for manganese inthe activation of a variety of Mn-dependent enzymes including pyruvatedehydrogenase phosphatase. Free manganese in the absence of calcium isnot able to activate the enzyme (see line 6 compared to line 2) However,the IPG Mn co-factor is able to stimulate activity in the absence ofcalcium (see line 4). The effect of co-factor binding is to make the PDHphosphatase responsive to calcium (see line 3). Release of the IPGtherefore activates the PDH phosphatase and the enzyme is now primed forfurther activation in the event calcium is also releasedintracellularly. In a homogolous fashion calcium is able to activate toPDH phosphatase (line 1) and the enzyme is now primed to respond to therelease of IPG (line 3). PDH phosphatase (activity) +Mg²⁺ + Ca²⁺ Δ 0.334OD/min (1) +Mg²⁺Ca²⁺ + Mn^(2+(sat)) Δ 0.680 OD/min (2) +Mg²⁺ + Ca²⁺ +IPG Δ 0.833 OD/min (3) +Mg²⁺ + IPG Δ 0.130 OD/min (4) +Mg²⁺ Δ 0.000OD/min (5) +Mg²⁺ + Mn^(2+(sat)) Δ 0.000 OD/min (6)

[0153] Glycogen Synthase Activity.

[0154] The results below indicate that the Mn²⁺ containing p-type IPGsstimulate glycogen synthase activity, in accordance with previousresults carried out on uncharacterised fractions from rat tissues.Incubation Stimulation as % of G6P Maximum No additions, +7.2 mM G6P 100Insulin 6.54 Placenta P-type (1) 2.82 Placenta P-type (2) 4.05

[0155] Placenta (1) was extracted using the equivalent of 5 g placentaand had been stored freeze-dried since tested before.

[0156] Placenta (2) was from the same extraction but was a mixture of 3samples of the same day in which the amount of tissue was 2.5, 1.0 and0.5 g but had been stored in solution at −20° C.

[0157] Effect on Lipogenesis.

[0158] The fraction containing the P-type IPG were added to ratadipocytes and the ability of these fraction to stimulate lipogenesisdetermined. Table 1 shows that no lipogenic activity was found in theP-type fraction from human liver.

[0159] Effects on NIH 3T3 Fibroblast Proliferation.

[0160] The P-type IPG fractions was assayed for its ability to supportproliferation of fibroblasts in the absence of serum. For rat tissuethis assay has been used to estimate the relative abundance of themediators since P-type mediators are active in the assay. The P-typefraction derived from human liver was found to be mitogenic when addedto fibroblasts transfected with the EGF receptor in serum-free media.FIG. 2 shows the dose-dependent effect obtained for the fraction.Saturation was not yet obtained at the highest concentrations used. Bothfractions however were able to induce proliferation at least 2-2.5 foldgreater than 10% FCS alone.

[0161] Descending Paper Chromatography.

[0162] A portion of the P-type material was treated with Pronase E for 2hours and then the pronase removed by acid precipitation. The solutionremaining was concentrated, redissolved in water and subjected topurification by descending paper chromatography usingn-butanol/ethanol/water. After development for 9 hours, the presence ofphosphate and free amino groups was detected. FIG. 3 shows thechromatogram profiles for the putative P-type mediator, followinganalysis for phosphate. Compounds containing phosphate were found tomigrate between the origin and 5 cm. The paper chromatograms were alsoanalysed for the presence of free amine groups as shown in FIGS. 3c andd. Again compounds containing free amino groups were present between theorigin and a migration distance of 5 cm. Incubation with pronase made nodifference to the migration of the compounds as assessed by thephosphate analysis as shown in FIGS. 3a and b.

[0163] Interaction with Ion Exchange Resins and Sep-Pak C18 Cartridges.

[0164] The behaviour of IPG in its interaction with two different ionexchange resins and a reverse phase C18 column were determined by theability of the eluates to induce thymidine incorporation into EGFTR17DNA cells. The results are shown in Table 2. In the case of the reversephase, 50-60% of the activity was recovered for the P-type IPG. Theseresults demonstrate that the P-type mediator is either a mixture ofhydrophilic and hydrophobic compounds both having proliferativeactivity, or the P-type mediators form some type of complex equilibriaresulting in partitioning depending on the physical state. Table 2 alsoshows that the P-type mediators could not be recovered from either acation exchange column (AG50-X12) or an anion exchange column (AG3-X4).This is consistent with the presence of dual functional groups such asfree amino and phosphate moieties as was found in FIG. 3.

[0165] Activity Requires Metal Ions.

[0166] The IPGs were extracted with dithizone (see Appendix 1, section2) to remove all metal ions. Following extraction, the P-type IPG wasinactive in the PDH phosphatase assay.

[0167] IPG Containing Carbohydrates.

[0168] Chromatograms of purified IPG P-type from human liver weredetected by pulse-amperimetic detections (conditions given in Appendix1, section 4).

[0169] The presence of various forms of inositol myo-inositol,chiro-inositol, pinitol) was confirmed using a DX500 system and a CarboPac MA1 column and pulsed ampherimetic detection (method given inAppendix 1, section 6).

[0170] Purification by High-Voltage Electrophoresis (HVE).

[0171] The material eluted from the paper after descendingchromatography was subjected to high-voltage paper electrophoresis at pH5.4. Under these conditions, negatively charged compounds containingphosphate, carboxy, or sulphate groups migrate towards the anode. Arepresentative paper electrophoretogram of three independent experimentsis shown in FIG. 4a following analysis for phosphate. Phosphate wasdetected at the origin and as a broad unresolved peak extending from 5cm to 20 cm migration distance. The profiles for the P-type mediatorswere remarkably similar. The presence of phosphate at the originindicates that compounds recovered in this position must have an equalnumber of positively charged moieties which neutralize the overallcharge. Those compounds which migrate either have an excess ofnegatively charged groups (e.g. phosphate) over positively chargedmoieties (e.g. amino, metal). FIG. 4b demonstrates that the cellproliferation activity of the putative P-type mediator is still presentafter the pronase treatment, paper chromatography and HVE purificationsteps. The activity profile following HVE very closely mirrors thephosphate analysis shown in FIG. 4a with activity present at the originand in a broad band extending to 20 cm migration distance. The fractionwas then assayed for its ability to stimulate PDH phosphatase and thisactivity correlates very strongly (R=0.97) with the ability of thefraction to stimulate cell proliferation as shown in FIG. 5a. FIG. 5bdemonstrates that there is a strong correlation (R=0.87) between thephosphate content of the putative P-type mediators isolated from thepaper electrophoretogram and the ability of some of the fractions takenfrom the electrophoretogram to stimulate cell proliferation. Thecorrelation between phosphate content and PDH phosphatase stimulatingactivity was also strong (R=0.73, data not shown).

[0172] The presence of hexoses and hexosamines was confirmed using a DX500 system and a Carbo Pac PA1 column and pulsed ampherimetic detection.

[0173] Vydac HPLC Chromatography

[0174] In order to demonstrate that it is possible to isolate and purifyP-type mediators from samples containing the family of compounds shownin FIG. 1, fractions obtained from a Vydac 301 PLX575 HPLC column andwere analysed for phosphate and growth promoting activity. FIG. 8 showsthe phosphate levels of the different fractions and FIG. 9 shows thebioactivity of the selected fractions including 7, 17, 25, 38 and 42.The predominant growth promoting activity was found in fractions 23-25.The Dionex HPLC profile of the main active fraction is shown in FIG. 10.This fraction contains predominantly peak 23 shown in FIGS. 1 and 7.

[0175] Dithizone Treatment of Liver P-type

[0176] Three samples of liver P-type used. All were suspended on day ofuse at the rate of 1 g liver in 10 μl water. All three preparations weretested before and after treatment with dithizone, while the onepreparation was also treated with Mn after dithizone to attempt toreactivate it. The reactivation procedure was to preincubate ade-metallized sample with 2.7 mM Mn for 15 min before it was added tothe inactivated PDH mix. No Treatment Vol used % stimulation Units/g  1.Original P-1 5 μl +58 2.32  2. Treated P-1 5 μl 0 0  3. 2.7 mM Mn 5 μl−11 0  4. Treated P-1 + 5 μl +30 1.20 2.7 mM Mn  5. Original P-2 5 μl+66 2.64  6. Treated P-2 5 μl +53 2.12  7. P-2 extracted 5 μl +91 3.64with chloroform  8. Water extracted 5 μl −2 0 with chloroform  9.Original P-3 5 μl +30 1.20 10. Treated P-3 5 μl +12 0.48

[0177] This experiment shows the following:

[0178] 1. Treatment with dithizone decreased activity of all threespecimens tried; P-1 by 100%, P-2 by 60% and P-3 by 20%.

[0179] 2. Preincubation with Mn restored approx half activity.

[0180] 3. Extraction of P-2 with chloroform alone enhances activity byalmost 40%. This may imply the presence of a chloroform solubleinhibitor.

[0181] 4. There is the anomaly that 10⁻⁴M Mn (assay no: 3 above) has noeffect on P′ase activity, whereas previous results clearly show thatthis concentration has a marked stimulatory action on P′ase.

[0182] Thus, removal of metal removes all activity from the IPG, butthat this activity can be reconstituted.

[0183] Effect of Mn and Zn on the Reactivation of Dithizone TreatedLiver P-Type IPG

[0184] In the last experiment Mn only partially reactivated P-type IPGthat had been stripped of its metal by treatment with dithizone. Thismight reflect that the IPG contains both Mn and Zn in the roughproportion of 3:2. The present experiment was to examine whether bothmetals activated and whether they were additive.

[0185] The P-1 sample used here was the same as that used in previousexperiment although a second tube of that composite sample. Thedithizone treated sample was the same as that used previously. Forreactivation, the de-medalist sample was pre-incubated for 15 mins witheither 2.7 mM Mn, 1.8 mM Zn or 2.7 mM Mn-1.8 mM Zn. All samples testedon normal assay system using 10 μl P′ase and 5 ul of sample. No Sample %stimulation 1. Original untreated P-1 +43 2. Treated P-1 0 3. P'ase +2.7 mm Mn +12 4. P'ase + 1.8 mM Zn +122 5. P'ase + Mn + Zn +111 6.Treated P-1 + Mn + Zn +262

[0186] This experiment showed the following:

[0187] 1. Zn stimulated P′ase to a greater extent than did Mn.

[0188] 2. The effect of Mn and Zn on P′ase was not additive.

[0189] 3. The effect of the combined metals on inactivated IPG was tostimulate the inactivated IPG to a value appreciably greater than couldbe expected from the sum of the original activity+Mn+Zn (43+12+122=177)to a value of 262.

[0190] Thus, this experiment confirms that the P-type IPG may require amixture of Mn²⁺ and Zn²⁺ for activity in some circumstances.

[0191] MALDI Mass Spectroscopy

[0192] High resolution MALDI mass spectrum (negative mode) of the familyof P-type molecules is shown in FIG. 11. The family of structure arerelated by the addition of 236 or 237 m/z structure units, see the threemarked peaks.

[0193] The molecular weights determined by negative mode MALDI massspectroscopy differ from the actual molecular weights of the P-typesubstances by the removal of a H⁺ atom, i.e. the actual weight can beobtained by adding +1 to the molecular weights of the peaks shown inFIG. 11. Thus, it is straightforward to determine the molecular weightsbased on the results in the figure.

[0194] Monoclonal Antibodies.

[0195] Inositolphosphoglycan (IPG) purified from rat liver by sequentialthin layer chromatography (TLC) was used to immunise New Zealand rabbitsand Balb/c mice by using conventional procedures.

[0196] After immunisation, monoclonal antibodies were prepared using theapproach of fusion of mouse splenocytes (5×10⁶ cells/ml) with mutantmyeloma cells (10⁶ cells/ml). The myeloma cell lines used were thoselacking hypoxanthine-guanine phosphoribasyl transferase. The screeningmethod of hybridoma cells was based on a non-competitive solid-phaseenzyme immunoassay in which the antigen (IPG) was immobilised on a solidphase. Culture supernatants were added and positive hybridoma cells wereselected.

[0197] A single cell cloning was made by limiting dilution. Hybridomasfor three monoclonal antibodies (2D1, 5HG and 2P7) were selected. Allmonoclonal antibodies were determined to be. IgM using a EK-5050 kit(Hyclone).

[0198] In order to test that all monoclonal antibodies recognised IPGs,a non-competitive solid-phase enzyme immunoassay was used. F96 PolysorpNunc-Immuno Plates are used for the assay. The polysorp surface isrecommended for assays where certain antigens are immobilised.

[0199] The immobilised antigen (IPG) diluted to 1:800 captured themonoclonal antibody from tissue culture supernatant, ascitic fluid, andwhen the purified monoclonal antibody was used.

[0200] The detection method used an anti-mouse IgM, biotinylated wholeantibody (from goat) and a streptavidin-biotinylated horseradishperoxidase complex (Amersham), ABTS and buffer for ABTS (BoehringerMannheim).

[0201] The same immunoassay was used to evaluate the polyclonalantibody. In this assay, the detection method employed an anti-rabbitIg, biotinylated species—specific whole antibody (from donkey).

[0202] The antibodies can be purified using the following method. FastProtein Liquid Chromatography (Pharmacia FPLC system) with a gradientprogrammer GP-250 Plus and high precision pump P-500 was used in orderto purify a polyclonal IPG antibody.

[0203] A HiTrap protein A affinity column was used for purification ofpolyclonal IPG from rabbit serum. Protein quantitation was made using aMicro BCA protein assay reagent kit (Pierce).

[0204] Monoclonal IgM antibodies were purified in two steps. Ammoniumsulfate precipitation was the method chosen as a first step. Tissueculture supernatant was treated with ammonium sulfate (50% saturation).Pellet diluted in PBS was transferred to dialysis tubing before thesecond step.

[0205] Since ammonium sulfate precipitation is not suitable for a singlestep purification, it was followed by gel filtrationchromatography-antibody solution in PBS run into a Pharmacia Sepharose4B column. Protein quantitation was made reading the absorbance at220-280 nm in a Perkin-Elmer lambda 2 UV/VIS spectrophotometer.

[0206] Thus, this example shows that it is possible to raise monoclonaland polyclonal antisera to the A and P-type substances. These could beused as antagonists or binding agents.

[0207] Discussion

[0208] Material isolated by elution from an AG1-X8 resin with 10 mM HCl(P-type IPG) stimulated pyruvate dehydrogenase phosphatase. Thisfraction also stimulated the proliferation of EGF-receptor transfected3T3 cells and glycogen synthesis in adipocytes.

[0209] The biological characteristics of the P-type IPG fractionisolated from human liver were recovered after treatment with pronase,indicating that its activity is not due to either protein or peptides.The presence of phosphate and free amino groups suggests that thesecompounds could be similar to those reported to contain hexoses andhexosamines in their structure. The strong correlations betweenphosphate content and the ability of the mediators to stimulate cellproliferation (FIG. 5a) and PDH phosphatase stimulating activity (FIG.5b) strongly suggests that phosphate is a key component of the putativemediators. The carbohydrate nature of these compounds is supported bytheir behaviour in descending paper chromatography, characteristic ofcarbohydrate-containing compounds and resembling that of the IPGisolated from insulin stimulated rat tissues. The Dionex profilesconfirm the presence of carbohydrate. All experiments were consistentwith the presence of P-type insulin-mimetic inositolphosphoglycans inthe 10 mM fraction eluted from the anion exchange resin.

[0210] In humans, post-receptor tissue insulin resistance of glucosemetabolism is a feature of non-insulin-dependent diabetes mellitus(NIDDM) and many other disorders. Resistance could result from anintrinsic defect in insulin signalling pathways or could be caused bythe presence of a circulating inhibitor of insulin action or both.Defects in IPG-associated mediator pathways therefore are key targetsfor investigations on the pathogenesis of NIDDM.

[0211] The importance of IPG in insulin signalling comes from both invitro and in vivo data. For example, mutant cells unable to make IPGrespond to insulin by tyrosine phosphorylation, but without metaboliceffects [1] and cells bearing kinase-deficient insulin receptors do nothydrolyse GPI following insulin stimulation [2]. There is also acorrelation with insulin receptor level with both insulin action andbreakdown of GPI [3]. The insulin resistance of cells from diabetic GKrats, which have a defect in GPI synthesis and release, can be overcomewith IPG from bovine liver [4]. Similarly antibody to an enzymaticallyand chemically modified inositol phosphate glycan isolated fromTrypanosome brucei blocks the effects of insulin [5,6,7,8] There isimpairment of insulin-stimulated hydrolysis of GPI in adipocytes fromstreptozotocin-diabetic rats and impaired insulin activation of pyruvatedehydrogenase (PDH) and glucose utilisation [5].

[0212] PDH activity of NZO mice is unresponsive to insulin stimulationin the presence of significant stimulation of glucose transport andutilisation, suggesting a post-receptor defect at the level of insulinstimulation of this enzyme. Insulin stimulates the production of P-typemediators (activates PDH) in lean NZC mouse adipocytes but paradoxicallycauses a decrease in mediator production or activity in adipocytes ofNZO mice [9]. No insulin antagonists are found. These resultsdemonstrate in NZO mice a post-receptor defect of insulin action at thelevel of pyruvate dehydrogenase activation (i.e. P-type mediator). Adefective mediator (or response to the mediator) has also been reportedin adipocytes of insulin-resistant, type II diabetic Goto-Kakizaki rats[10] The decreased urinary chiro-inositol (hydrolysis product of P-typemediator) secretion found in patients with type II diabetes [11,12]suggests a similar post-receptor defect in human patients, although notall studies have been able to confirm these observations [13].

[0213] There is decreased urinary chiro-inositol in spontaneouslydiabetic (fat) rhesus monkeys [14], and chiro-inositol lowers plasmaglucose in such monkeys and in streptozotocin-treated rats, andactivates glycogen synthase [15]. Intravenous infusion of the mediatorsin streptozotocin-treated rats decreases plasma glucose without a changein the serum insulin concentrations and ip injection results inglycogenic changes in diaphragm.

REFERENCES

[0214] The contents of all of the references listed below or mentionedin the description above are incorporated herein by reference.

[0215] 1. Lazar-DF, Knez J J, Medof-M E, Cuatrecasas-P, Saltiel-A R.1994 Stimulation of glycogen synthesis by insulin in humanerythroleukemia cells requires the synthesis ofglycosyl-phosphatidylinositol. Proc Natl Acad Sci USA. 91: 9665-9669.

[0216] 2. Villalba M, Alvarez J F, Russell D S, Mato J M, Rosen O M.1990 Hydrolysis of glycosyl-pnosphatidylinositol in response to insulinis reduced in cells bearing kinase-deficient insulin receptors. GrowthFactors. 2: 91-97.

[0217] 3. Macaulay S L, Clark S, Larkins R G. 1992 Correlation ofinsulin receptor level with both insulin action and breakdown of apotential insulin mediator precursor: studies in CHO cell-linestransfected with insulin receptor cDNA. Biochim Biphys Acta. 1134:53-60.

[0218] 4. Farese R V, Standaert M L, Yamada K, Huang L C, Zhang C,Cooper D R, Wang Z, Yang Y, Suzuki S, Toyota T, Larner J. 1994Insulin-induced activation of glycerol-3-phosphate acyltransferase by achiro-inositol-containing insulin mediator is defective in adipocytes ofinsulin-resistant type II diabetic, Goto-Kakizaki rats. Proc Natl AcadSci USA. 91: 11040-11044.

[0219] 5. Huang L C, Fonteles M C, Houston D B, Zhang C, Larner J. 1993Chiroinositol deficiency and insulin resistance. III. Acute glycogenicand hypoglycemic effects of two inositol phosphoglycan insulin mediatorsin normal and streptozotocin-diabetic rats in vivo. Endocrinology. 132:652-657.

[0220] 6. Nestler J E, Romero G, Huang L C, Zhang C G, Larner J. 1991Insulin mediators are he signal transduction system responsible forinsulin's actions on human placental steroidogenesis. Endocrinology.129: 2951-2956.

[0221] 7. Romero G, Gamez G, Huang L C, Lilley K, Luttrell L. 1990Anti-inositolglycan antibodies selectively block some of the actions ofinsulin in intact BC3H1 cells. Proc Natl Acad Sci USA. 87: 1476-1480.

[0222] 8. Represa J, Avila M A, Miner C, Giraldez F. Romero G, ClementeR, Mato J M, Varela-Nieto I. 1991 Glycosyl-phosphatidylinositol/inositolPhosphoglycan: a signaling system for the low-affinity nerve growthfactor receptor. Proc Natl Acad Sci USA. 88: 8016-8019.

[0223] 9. Macaulay S L, Larkins R G. 1988 Impaired insulin action inadipocytes of New Zealand obese mice: a role for postbinding defects inpyruvate dehydrogenase and insulin mediator activity. Metabolism. 37:958-965.

[0224] 10. Farese R V, Standaert M L, Yamada K, Huang L C, Zhang C,Cooper D R, Wang Z, Yang Y, Suzuki S, Toyota T, et al. 1994 Insulininduced activation of glycerol-3-phosphate acyltransferase by achiro-inositol-containing insulin mediator is defective in adipocytes ofinsulin-resistant, type II diabetic, Goto-Kakizaki rats. Proc. Natl,Acad. Sci. U.S.A. 91: 11040-4.

[0225] 11. Kennington A S, Hill C R, Craig J, Bogardus C, Raz I,Ortmeyer H K, Hansen B C, Romero G, Larner J. 1990 Low urinarychiro-inositol excretion in non-insulin-dependent diabetes mellitus. NEngl J. Med. 323: 373-378.

[0226] 12. Suzuki S, Kawasaki H, Satoh Y, Obtomo M, Eiriai M, Hirai A,Hirai S, Onoda M, Matsumoto M, Hinokio Y. et al. 1994 Urinarychiro-inositol excretion is an index marker of insulin sensitivity inJapanese type II diabetes. Diabetes-Care. 1994 17: 1465-8.

[0227] 13. Ostlund R E Jr, McGill J B, Herskowitz I, Kipnis D M,Santiago J V, Sherman W R 1993 D-chiro-inositol Metabolism in diabetesmellitus. Proc. Natl. Acad. Sci. U.S.A. 1993 90: 9988-92.

[0228] 14. Ortmeyer H K, Bodkin N L, Lillley K, Larner J, Hansen B C.1993 Chiroinositol deficiency and insulin resistance I. Urinarysecretion rate of chiroinositol is directly associated with insulinresistance in spontabeously diabetic rhesus monkeys. Endocrinology. 132:640-645.

[0229] 15. Ortmeyer H K, Huang L C, Zhang L, Hansen B C, Larner J. 1993Chiroinositol deficiency and insulin resistance. II. Acute effects ofD-chiroinositol administration in streptozotocin-diabetic rats, normalrats given a glucose load, and spontaneously insulin-resistant rhesusmonkeys. Endocrinology. 132: 646-651.

What is claimed is:
 1. A monoclonal antibody that specifically binds to an isolated P-type substance obtainable from human liver or placenta, wherein the substance is a cyclitol-containing carbohydrate comprising Mn²⁺ and Zn²⁺ ions and has the biological activity of activating pyruvate dehydrogenase (PDH) phosphatase.
 2. The monoclonal antibody of claim 1 wherein the substance comprises phosphate.
 3. A hybridoma producing the monoclonal antibody of claim
 1. 4. A pharmaceutical composition comprising the monoclonal antibody of claim 1 in combination with a pharmaceutically acceptable carrier.
 5. The monoclonal antibody of claim 1, wherein the monoclonal antibody is an antagonist having the property of: a) inhibiting the release of the P-type substance; b) binding to the P-type substance and thereby reducing its level; and/or c) reducing a biological activity of the P-type substance.
 6. The monoclonal antibody of claim 1, wherein the monoclonal antibody is linked, directly or indirectly, to a label.
 7. The monoclonal antibody of claim 1, wherein the monoclonal antibody is immobilized on a solid phase.
 8. An immunoassay method comprising: a) contacting a biological sample with the monoclonal antibody of claim 1 under suitable conditions for specific binding of the monoclonal antibody to P-type substance present in the sample, if any; and b) determining whether the monoclonal antibody binds specifically to the sample.
 9. The immunoassay method of claim 8, additionally comprising measuring the amount of specific binding as an indication of the concentration of the P-type substance in the sample.
 10. The immunoassay method of claim 9, additionally comprising determining the concentration of one or more A-type inositolphosphoglycans (IPGs) and then determining the ratio of the concentration of the P-type substance determined in the immunoassay method to the concentration of A-type IPG(s).
 11. A monoclonal antibody that specifically binds to a P-type cyclitol-containing carbohydrate substance comprising Mn²⁺ and Zn²⁺ ions, wherein the substance has the biological activity of activating pyruvate dehydrogenase (PDH) phosphatase and a molecular weight determined using negative mode MALDI mass spectroscopy as shown in FIG. 11, or a molecular weight related to one of the molecular weights set out in FIG. 11 by the addition or subtraction of one or more structure units of about 236 m/z.
 12. The monoclonal antibody of claim 11 wherein the substance comprises phosphate.
 13. A hybridoma producing the monoclonal antibody of claim
 11. 14. A pharmaceutical composition comprising the monoclonal antibody of claim 11 in combination with a pharmaceutically acceptable carrier.
 15. The monoclonal antibody of claim 11, wherein the monoclonal antibody is an antagonist having the property of: a) inhibiting the release of the P-type substance; b) binding to the P-type substance and thereby reducing its level; and/or c) reducing a biological activity of the P-type substance.
 16. The monoclonal antibody of claim 11, wherein the monoclonal antibody is linked, directly or indirectly, to a label.
 17. The monoclonal antibody of claim 11, wherein the monoclonal antibody is immobilized on a solid phase.
 18. An immunoassay method comprising: a) contacting a biological sample with the monoclonal antibody of claim 11 under suitable conditions for specific binding of the monoclonal antibody to P-type substance present in the sample, if any; and b) determining whether the monoclonal antibody binds specifically to the sample.
 19. The immunoassay method of claim 18, additionally comprising measuring the amount of specific binding as an indication of the concentration of the P-type substance in the sample.
 20. The immunoassay method of claim 19, additionally comprising determining the concentration of one or more A-type inositolphosphoglycans (IPGs) and then determining the ratio of the concentration of the P-type substance determined in the immunoassay method to the concentration of A-type IPG(s). 